118 Chapter 5 of DNA methylation assays in service laboratories that have a cervical screening and/ or diagnostic remit. Clinician-collected cervical samples are taken under relatively standardised conditions and in that way likely well suited for protocols with standardised input volume, as is also the case in many HPV test platforms. With the introduction of more sample-to-result HPV assays a direct cell conversion protocol is highly beneficial as a physical DNA isolate may not be generated as a result of the standard HPV testing workflow in cervical screening. Automated protocols could further improve efficiency and considerably enhance the practicability of methylation analysis in diagnostic and FAM19A4 (A) Direct cell conversion protocol (ΔΔCq value) 2 6 10 14 18 6 2 10 14 18 Reference protocol (ΔΔCq value) Bland-Altman plot of FAM19A4 (C) Difference between the direct cell conversion protocol and the reference protocol 2.5 5.0 7.5 10.0 15.0 12.5 -2 -4 0 2 4 Average of the two protocols (ΔΔCq value) Bland-Altman plot of miR124-2 (D) Difference between the direct cell conversion protocol and the reference protocol 2.5 5.0 7.5 10.0 15.0 12.5 -2 -4 0 2 4 Average of the two protocols (ΔΔCq value) Control CIN2 CIN3 miR124-2 (B) Direct cell conversion protocol (ΔΔCq value) 2 6 10 14 18 6 2 10 14 18 Reference protocol (ΔΔCq value) Figure 5.2 Correlation of ΔΔCq values of FAM19A4 (A) and miR124-2 (B) between direct cell conversion and reference protocol. For both x- and y-axis: a lower ΔΔCq value represents a higher methylation value. Bland-Altman plot for FAM19A4 (C) and miR124-2 (D). The horizontal red line shows the mean of the difference in ΔΔCq value (=bias) between the two protocols, being 0.167 for FAM19A4 and -0.142 for miR124-2. The horizontal green lines show the upper and lower 95% limits of agreement (= bias ±1.96 × SD), being -2.274 and +2.608, respectively, for FAM19A4 and -2.316 and +2.031, respectively, for miR124-2. Abbreviations: Cq, quantification cycle; CIN, cervical intraepithelial neoplasia
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